Method for reducing amino acid biosynthesis inhibiting effects of a sulfonyl-urea based compound

ABSTRACT

A method has been found for reducing amino acid biosynthesis inhibiting effect of a sulfonyl-urea based compound of the general formula:                    
     comprising contacting in an aqueous solution said sulfonyl-urea based compound with an enzyme capable of reducing said amino acid biosynthesis inhibiting effect.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. 119 of Danishapplication PA 1998 01325 filed Oct. 15, 1998 and of U.S. Provisionalapplication No. 60/105,146 filed Oct. 21, 1998, the contents of whichare fully incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a method for enzymatic treatment ofsulfonyl-urea based compounds. More specifically the invention relatesto a method, wherein an enzyme is employed to treat sulfonyl-urea basedcompounds, which are highly potent inhibitors of biosynthesis of aminoacids in various organisms, and which may be used as powerfulherbicides, pesticides or as drugs. Even more specifically the inventionrelates to a method for reducing the amino acid biosynthesis inhibitingeffect of sulfonyl-urea based compounds to prevent unintendedcontamination of biological material sensitive to such compounds is bydevices used at both sensitive and insensitive biological material.

BACKGROUND ART

Sulfonyl-urea based compounds are known to the art as powerfulinhibitors of amino acid biosynthesis, which thus may be used to inhibitthe proliferation of cells or organisms or even kill them. In theagricultural and horticultural area sulfonyl-urea based compounds, suchas Tribenuron ™ and Metsulforon ™ are, inter alia, known as highlypotent herbicides. Although the high amino acid biosynthesis inhibitingeffect of these compounds allows for a desired general reduction in theload on the locus to which it is administered, these compounds mayhowever by nature be harmful to other biological entities ifcontaminated.

The high amino acid biosynthesis inhibiting effect of sulfonyl-ureabased compounds makes it critical to avoid that even trace amounts ofthe active compounds contaminate biological material where the compoundsmay have a harmful effect. Thus it is highly desired to reduce the riskof contamination, by reducing amino acid biosynthesis inhibiting effectof remaining amounts of sulfonyl-urea based compounds present e.g. indevices which is used both to administer these compounds to the intendedbiological locus and to loci where even trace amounts of thesulfonyl-urea based compounds have a harmful effect.

SUMMARY OF THE INVENTION

We have found that the amino acid biosynthesis inhibiting effect ofsulfonyl-urea based compounds may be reduced or even eliminated bytreating these compounds with an enzyme. Accordingly we have found amethod for reducing amino acid biosynthesis inhibiting effect of asulfonyl-urea based compound of the general formula:

comprising contacting in an aqueous solution said sulfonyl-urea basedcompound with an enzyme.

DETAILED DISCLOSURE OF THE INVENTION

Definitions

The amino acid biosynthesis inhibiting effect as used herein inaccordance with the invention is to be construed as the amount ofcompound calculated in an unambiguous unit such as concentration ormoles required to obtain a predefined effect in a process, to which thecompound is applied. Accordingly a compound which has a high amino acidbiosynthesis inhibiting effect in a predefined process will require lessconcentration or amount of the compound than a compound with less aminoacid biosynthesis inhibiting effect to obtain the same effect. Aminoacid biosynthesis inhibiting effect may be evaluated as an absoluteproperty of a compound by measuring the absolute effects of the compoundin a process at predefined conditions or it may be evaluated as arelative property of a compound, whereby the effect of the compound ismeasured in a process and compared to a reference compound measured inthe same process. As an example, the compound may be a herbicide and theprocess may be the killing or growth inhibition of a specific herb in afield. The amino acid biosynthesis inhibiting effect of a compound maythus be evaluated by spraying half the field with a solution of thecompound in a specific ratio (e.g. liters per square acre) and the otherhalf with an equally concentrated solution of a reference herbicide. Theamino acid biosynthesis inhibiting effect of the compound may then beevaluated as the number of herbal plants killed or growth inhibited bythe compound compared to the number of herbal plants killed or growthinhibited by the reference herbicide in each half of the field.

The enzyme

The enzyme nomenclature used herein is based on the recommendations ofthe Nomenclature Committee of the International Union of Biochemistryand Molecular Biology (IUBMB) which describes types of characterizedenzymes with an EC (Enzyme Commission) number.

Enzymes suitable to be employed in the method of the invention may inprinciple be any enzyme which is able of reducing the amino acidbiosynthesis inhibiting effect of a sulfonyl-urea based compound.Preferably the enzyme has been industrially produced by fermentation, byin vitro synthesis or by extraction from plant or animal tissue, andmore preferably the enzyme has been isolated or purified by methodsknown to the art. A variety of enzyme types may provide a reduction inamino acid biosynthesis inhibiting effect, each in different ways.

Preferred enzymes are hydrolases within the enzyme class of EC 3.-.-.-,such as enzymes from the subclasses: esterases (EC 3.1.-.-), peptidases(EC 3.4.-.-), hydrolases acting on carbon-nitrogen bonds other thanpeptide bonds (EC 3.5.-.-) and sulfohydrolases (EC 3.10.-.-).

Among esterases suitable enzymes are carboxylic ester hydrolases (EC3.1.1.-), such as carboxyl esterase (EC 3.1.1.1) and lipase (EC3.1.1.3), or sulphatases (EC 3.1.6.-), while suitable hydrolases actingon carbon-nitrogen bonds other than peptide bonds are amidases (EC3.5.1.4) and ureases (EC 3.5.1.5) such as urease obtained from Jackbeans, e.g. Sigma (art. no. U 2125).

The enzyme used in the method of the invention may be synthesized invitro or it may preferably be obtained by culturing a microorganismexpressing the mature or immature enzyme in a suitable medium andrecovering the enzyme by methods known to the art. The enzymes may beobtainable from a variety of microbial sources, notably bacteria andfungi (including filamentous fungi and yeasts). The enzymes may evenmore preferably be obtained by

isolating a DNA sequence encoding the desired enzyme from a microbialsource,

inserting said DNA sequence in a functional expression vector byrecombinant techniques known to the art,

Inserting the functional expression vector in a microbial host cell(e.g. by transformation),

Culturing the transformed host cell in a medium suitable for expressionof the enzyme.

Recovery and formulation of the enzyme.

The fermentation broth or enzyme solution or concentrates thereof may befurther processed to obtain 1) a stable liquid composition by additionof conventional stabilizers, 2) a slurry composition or 3) a compositionwith the enzyme in a protected form. Protected enzymes may be preparedaccording to the method disclosed in EP-A-238,216.

Solid enzyme preparations or composition may be prepared from the brothor enzyme solution or concentrates thereof broth by precipitation withsalts, such as Na₂SO₄ or water-miscible solvents, such as ethanol oracetone. Removal of the water in the broth by suitable drying methods,such as spray-drying, may also be employed. A preferred solid enzymecomposition is a granulate, most preferred a dust free granulate. Dustfree granulates may be produced, e.g. as disclosed in U.S. Pat. No.4,106,991 and U.S. Pat. No. 4,661,452 and may optionally be coated bymethods known to the art.

The Sulfonyl-urea Based Compound

The sulfonyl-urea based compounds are of the general formula:

where R1 and R4 may be selected from the group of residues consisting ofC₁₋₁₈-alkyl, monocyclic aromatic, dicyclic aromatic, polycyclic aromaticand heteroaromatic, while R2 and R3 substituent may be selected from thegroup consisting of hydrogen, methyl, ethyl and butyl. in a preferredembodiment R1 is a mono-aromatic group and R4 is a heteroaromatic group,e.g. R1 may be a phenyl group and R4 may be selected from the groupconsisting of pyridine, pyrazine, pyridazine, pyrimidine and triazine.Thus the a preferred sulfonyl-urea based compound may be of the formula:

Said R1 and R4 substituents may further be substituted with substituentsselected from the group consisting of halogen, sulfo, sulfonato,sulfamino, sulfanyl, amino, amido, nitro, azo, imino, carboxy, cyano,formyl, hydroxy, halocarbonyl, carbamoyl, carbamidoyl, phosphonato,phosphonyl, C₁₋₁₈-alkyl, C₁₋₁₈-alkenyl, C₁₋₁₈-alkynyl, C₁₋₁₈-alkoxy,C₁₋₁₈-oxycarbonyl, C₁₋₁₈-oxoalkyl, C₁₋₁₈-alkyl sulfanyl, C₁₋₁₈-alkylsulfonyl and C₁₋₁₈-alkyl imino or amino which is substituted with one,two or three C₁₋₁₈-alkyl groups. Accordingly the sulfonyl-urea basedcompound may preferably be selected from the group consisting of

Further preferred sulfonyl-urea based compounds are biocides such as aherbicide, pesticide, fungicide or bactericide and preferably acts asamino acid biosynthesis inhibitors.

Applications

The method of the invention comprises as said contacting in an aqueoussolution the sulfonyl-urea based compound with an enzyme capable ofreducing the amino acid biosynthesis inhibiting effect of thesulfonyl-urea based compound. Typically the reduction of amino acidbiosynthesis inhibiting effect may be obtained from contact with ahydrolase enzyme which will result from a hydrolytic degradation ordigestion of the sulfonyl-urea based compound.

The method may suitably be used to clean devices used for handling thecompounds, which devices are also used for purposes where the presenceof sulfonyl-urea based compounds is highly undesirable. Such devices maye.g. be agricultural or horticultural crop spraying devices used both tospray plants, which is insensible to sulfonyl-urea based compounds andplants which is sensible to sulfonyl-urea based compounds. The methodmay also be used to avoid general environmental contamination bytreating e.g. left overs of spraying solutions comprising sulfonyl-ureabased compounds, and waste waters used to clean or flush the sprayingdevice. Accordingly the invention provides a method for cleaning aspraying device or a waste water containing sulfonyl-urea basedcompounds by contacting the spraying devices or waste water with anenzyme.

Materials and Methods

All chemicals should be of analytical grade and may be obtained fromMerck (Darmstadt, Germany). Chlorsulfuron(1-(2-chlorophenylsulfonyl)-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea)is available from E.I. du Pont de Nemours & Co. (Inc.) under thetrademarks Glean and Telar.

As a method of measuring the Glean and Telar and the degradationproducts upon treatment with enzyme, HPLC is performed using a LC-6AHPLC system (Shimadzu, Japan) with UV-detection (280 nm), employing agradient of acetonitrile in water (20-80% over 25 min.) and a LiChrosorbRP-18 (10 μm) reversed phase column (250×4.6 mm I.D.).

A second assay for detecting levels of sulfonyl-urea based compounds insamples was developed in which 2.25 mg chorsulfuron was dissolved in a1500 μl BR buffer pH 7, which has been added 125 μl acetone and 125 μlethanol. The BR buffer was 40 mM phosphoric acid, 40 mM acetic acid, 40mM boric acid adjusted with 0.2N NaOH. Samples of typically 20 μl ofenzyme stock solution (dependent on the concentration) was added to thismixture and the resulting mixture was incubated at 37° C. for 24 hoursin a thermo mixer after which break-down of chorsulfuron could bemonitored by Thin Layer Chromatography (TLC) employing Si-60 plateseluted with a solvent of toluene, methylenechloride and ethanol in aratio of 5:4:1 (R_(f) values: chlorsulfuron 0.37, methylsyringate 0.51).

Urease obtained from Jack Beans (Type VI) obtained from Sigma (art. No.U 2125) is used to degrade the Chlorsulfuron(1-(2-chlorophenylsulfonyl)-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea).

The invention is illustrated by the following examples, which in no wayis intended to be limiting to the scope of the invention.

EXAMPLES Example 1

Degradation of Chlorsulfuron Using Urease

1 mg Urease (corresponding to approx. 125 Urease units) is added to asolution of 100 mg chlorosulfuron in 500 ml phosphate buffer (0.1M, pH7.0). The solution is stirred on a magnetic stirrer and samples aredrawn at regular time intervals, e.g. 1-5 minutes for 30 minutes andanalyzed by HPLC to monitor the sample content of chlorosulfuron. Acontrol sample prepared without the addition of Urease chlorosulfuron isalso analyzed by HPLC for comparison and relative measurement of thedegree of the enzymatic degradation of the chlorosulfuron.

What is claimed is:
 1. A method for reducing the amino acid biosynthesisinhibiting effect of a sulfonyl-urea based compound of the generalformula:

comprising contacting in an aqueous solution said sulfonyl-urea basedcompound with an isolated enzyme.
 2. The method according to claim 1,further characterized in that the enzyme is a hydrolase (EC 3.-.-.-). 3.The method according to claim 2, wherein said hydrolase is selected fromthe group consisting of esterase (EC 3.1.-.-), peptidase (EC 3.4.-.-),hydrolase acting on carbon-nitrogen bonds other than peptide bonds (EC3.5.-.-) and sulfohydrolase (EC 3.10.-.-).
 4. The method according toclaim 3, wherein said esterase is selected from the group consisting ofcarboxylic ester hydrolase (EC 3.1.1.-) and sulphatase (EC 3.1.6.-). 5.The method according to claim 4, wherein said carboxylic ester hydrolaseis selected from the group consisting of carboxyl esterase (EC 3.1.1.1)and lipase (EC 3.1.1.3).
 6. The method according to claim 3, whereinsaid hydrolase acting on carbon-nitrogen bonds other than peptide bondis selected from the group consisting of amidase (EC 3.5.1.4) and urease(EC 3.5.1.5).
 7. The method according to claim 6, wherein said urease isobtained from Jack Beans.
 8. The method according to claim 1, wherein R1and R4 of said general formula for the sulfonyl-urea based compound isselected from the group of residues consisting of C₁₋₁₈-alkyl,monocyclic aromatic, dicyclic aromatic, polycyclic aromatic andheteroaromatic and R2 and R3 is selected from the group consisting ofhydrogen, methyl, ethyl and butyl.
 9. The method according to claim 8,wherein R1 is a mono-aromatic group and R4 is a heteroaromatic group.10. The method according to claim 9, wherein said mono-aromatic group isa phenyl group and said heteroaromatic group is selected from the groupconsisting of pyridine, pyrazine, pyridazine, pyrimidine and triazine.11. The method according to claim 10, wherein the sulfonyl-urea basedcompound is of the formula:


12. The method according to claim 8, wherein said R1 and R4 substituentsfurther is substituted with substituents selected from the groupconsisting of halogen, sulfo, sulfonato, sulfamino, sulfanyl, amino,amido, nitro, azo, imino, carboxy, cyano, formyl, hydroxy, halocarbonyl,carbamoyl, carbamidoyl, phosphonato, phosphonyl, C₁₋₁₈-alkyl,C₁₋₁₈-alkenyl, C₁₋₁₈-alkynyl, C₁₋₁₈-alkoxy, C₁₋₁₈-oxycarbonyl,C₁₋₁₈-oxoalkyl, C₁₋₁₈-alkyl sulfanyl, C₁₋₁₈-alkyl sulfonyl andC₁₋₁₈-alkyl imino or amino which is substituted with one, two or threeC₁₋₁₈-alkyl groups.
 13. The method according to claim 8, wherein saidsulfonyl-urea based compound is selected from the group of compoundhaving the formulas of:


14. The method according to claim 1, wherein the sulfonyl-urea basedcompound is present in a spraying device or a waste water.